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that agent and the charge characteristics of the gel-ligand combination. Is it anionic, cationic, or neutral? Is it temperature sensitive? Is it subject to degradation by enzymatic action? To maintain the same level of activity after storage as existed previously, choose a bacteriostatic agent that will not bind to the gel matrix or ligand, and one that is easily washed out when the gels are reused (e.g., ethanol). Carefully remove all of the storage solution prior to reuse to prevent denaturation of the sample. Do not freeze the gel at any time. This will disrupt the matrix and can lead to fine particles that can interfere with the buffer flow. Again, follow the manufacturer's advice for proper storage.

[30] A f f i n i t y C h r o m a t o g r a p h y : S p e c i a l i z e d T e c h n i q u e s

By STEVEN OSTROVE and SHELLY WEISS This chapter discusses some specialized affinity chromatography techniques: cell affinity chromatography, metal chelate affinity chromatography, covalent affinity chromatography, and other binding techniques and the scaling up of affinity chromatography. It will be a guide in the use of these techniques and give a start in understanding the reasons behind their use. In addition, some of the possible problems and danger areas associated with these techniques are described. Not all of the specific methodologies available for separation by affinity chromatography will be reviewed in this chapter, nor will it provide an exhaustive list of examples for each technique. As you read this chapter, and try to use the techniques, however, you will find new and different ways to accomplish your separation task. Certain assumptions need to be made before we begin: First, that you are aware of general affinity chromatography procedures; second, that you know how some parameters such as temperature, pH, ionic strength, and flow rates affect affinity separations (see [29] in this volume). Cell Affinity Chromatography Isolating cells by affinity chromatography requires some special considerations due to the size and sensitivities of the living cell. Cells can be separated by affinity chromatography in two ways: either by binding the cell directly to the matrix as one binds a protein, or by binding a protein or METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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other ligand that can "recognize" a specific protein or receptor on the cell membrane. The second method is more commonly used. Cells are considerably larger than proteins, and can be even larger than the average bead size used for separating proteins. For this chapter we will assume an average cell size of 50-70/zm. The bead size used as the chromatographic support must be large enough to allow the passage of the cells between the beads when they are packed in a column. A bead diameter of 250-350/zm is usually adequate. The bed support (net) in the column must also be of a size that allows passage of the cells from the column. In general, the mesh size of this bed support should be at least 80 /zm (larger than the cells, but smaller than the matrix beads). This allows the cells to pass through without any significant impediment. The matrix must exhibit all the characteristics of a good affinity support (see [29] in this volume) and in addition must be nontoxic to the cells if they are to remain viable. One additional requirement for the resin used to separate cells is the ability to withstand sterilization either by autoclaving or chemical treatment. Cells have many sites on their membrane that can be utilized for their separation. For example, glycoproteins, which are common membrane components, may be selected by using the appropriate lectins. For example, concanavalin A (ConA) will select those proteins containing glucose or mannose, while wheat germ lectin will select those proteins containing N-acetylglucosamine. Other compounds, such as protein A, which binds to the Fc portion of immunoglobulins, can select cells with antibodies on their surface. Consequently, the choice of the affinity ligand is dependent on the cell type being purified. Cells can also be bound directly to the matrix through coupling agents such as cyanogen bromide (CNBr). This reagent binds to amines (preferentially to primary amines) found on cell surface proteins. The process of binding cells to ligands attached to a matrix is very similar to other affinity purifications. The procedure is summarized as follows: 1. 2. 3. 4. 5. 6.

Prepare matrix. Wash matrix-ligand complex. Slowly add cell suspension. Wash out nonadhering cells. Add elution buffer (specificity is important). Collect cells.

Elution of cells should be accomplished using a specific eluent for the ligand-cell (protein) complex since salt gradients are not recommended due to their osmotic effects on the cells. Special attention should be given

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to controlling pH, osmotic pressure, temperature, shear forces, and sterility. The necessary degree of control depends on the planned use of the cells following their separation. Flow rates for processing cells must also be adjusted so that they do not rupture. Shear forces can act on the cells as they pass through the matrix, causing changes in membrane structure or even some change in cell biochemistry. Thus, there may be alterations in one or more of the cells biochemical pathways as it "adjusts" to the stress of chromatography. For this reason flow rates in cell affinity chromatography are usually slower than in other affinity chromatography procedures. If, after separation, the purified cells are to be grown in culture, then the whole process must be done under aseptic conditions. The ligand, the matrix, and all buffers must be sterilized prior to use. Sterile conditions are not as important if affinity purification is the last step in the process, or if the cells will be used in short-term cultures (those lasting only a few hours), which do not require aseptic handling. Chelation Affinity Chromatography Immobilized metal affinity chromatography (IMAC), 1 also known as metal chelate affinity chromatography (MCAC), is a specialized aspect of affinity chromatography applicable to a wide variety of compounds. It was developed over a decade ago by Porath e t al. 2 as a novel approach to protein fractionation. Over the years it has increased in acceptance as a quick, reliable separation technique. At this time, however, its potential has not been fully explored. The principle behind IMAC lies in the fact that many transition metal ions, i.e., zinc 3 and copper, can coordinate to the amino acids histidine, cysteine, and tryptophan via electron donor groups on the amino acid side chains. In order to utilize this interaction for chromatographic purposes, the metal ion must be immobilized onto an insoluble support. This can be done by attaching a chelating group to the chromatographic matrix. Most importantly, in order to be useful, the metal of choice must have a higher affinity for the matrix than for the compounds to be purified. The most common chelating group used in this technique is iminodiacetic acid (IDA). It is coupled to a matrix such as Sepharose 6B, via a long hydrophilic spacer arm. The spacer arm ensures that the chelating metal is fully accessible to all available binding sites on a protein. Another popular chelating group for IMAC applications is tris(carboxymethyl)i j. Porath and B. Olin, Biochemistry 22, 1621 (1983). 2 j. Porath, J. Carlsson, and I. Olsson et al., Nature (London) 258, 598 (1975). 3 D. C. Rijken and D. Collen, J. Biol. Chem. 256, 7035 (1981).

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ethylenediamine (TED). This particular group lends different properties to the gel than IDA. TED gels show stronger retention of metal ions and weaker retention of proteins relative to that of IDA gels. TED gels form a complex (single coordination site) vs a chelate (multiple coordination sites) for IDA gels. The most commonly used metals for IMAC are zinc and copper; however, nickel, cobalt, and calcium have also been used successfully. Theoretically, other heavy metals and transition elements can be utilized, but there is a shortage of information in this area. The basic methodology of IMAC is quite simple. There are three main steps: (1) Charging the gel, (2) binding the proteins, and (3) eluting the proteins. Charging the IMAC gel usually involves passing a solution of the metal salt (ZnCI2 or CuSO4.5H20) over a column packed with the uncharged chelating matrix. The choice of the best metal is not always predictable. Copper often affords much tighter binding to proteins then does zinc. However, the weaker binding achieved using zinc may be a useful factor in some cases. Unless there are previous data, the appropriate choice of metal is a trial and error process. As with other affinity chromatographic techniques it is not generally recommended to use the full capacity of the gel for the metal, but to use one-third to one-half of the gel's capacity. This is particularly relevant when extremely strong binding of the protein occurs.

The most important factor affecting protein binding is the pH. Most protein binding will occur in the range of pH 6-8. At more alkaline pH values binding will most likely be via deprotonated amino groups. The choice of binding buffer is also critical. Avoid buffers containing any type of chelating agent such as EDTA or citrate. Tris, phosphate, and acetate buffers are all suitable for the pH range used for binding. Tris-HC1 (but not acetate) may reduce binding 4 and should be used only when the metal-protein affinity is quite high. Additional reagents such as urea, salts, or detergents may be added to the binding buffer with either little or no effect on binding. Porath and Olin I have explored this area in detail. Generally accepted, however, is that high concentrations of salt should be present to quench any ionexchange effect. Usually a concentration range of 0.5 to 1 M NaC1 is sufficient. Several methods can be employed for elution of biomolecules from a metal chelate affinity column. Each has advantages and the best method for any given separation must be experimentally determined. 4 C. A. K. Borrebaeck et al., F E B S Lett. 130, 194 (1981).

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Since binding is a pH-dependent function, a pH gradient is the most common method of elution. At the pH required for binding (pH 6 to 8) the groups which coordinate with the metal are deprotonated. Use of a decreasing pH gradient, i.e., pH 7 to 6, will cause protonation and subsequent elution. An alternative elution protocol is the use of a competitive ligand. In the case of IMAC, this involves increasing gradients of imidazole, histamine, glycine, or ammonium chloride. As with other affinity elutions that use competitive ligands, it is best accomplished at a constant pH, and usually the sample application buffer is used. Another elution method is the use of a chelating agent such as EDTA or EGTA. In this case, the metal ions will be completely stripped from the matrix. This may be useful for a quick group-specific elution. However, the disadvantage is that all adsorbed biomolecules will come off together. There will be no resolution of different species. IMAC was first utilized for separating serum proteins. 2 Many of the major serum proteins have an affinity for metal ions. Different proteins can be isolated with different chelate columns, sometimes used in series. Presently, there are many applications for this technique, and some of them are listed in Table I. 5-1° Interferons have been the subject of intense study for many years. A number of different mammalian interferon species have been purified by IMAC. T M Furthermore, Suikowski e t al. 17 have used this technique to study the surface topography of various interferon species since the affinity for the metal is dependent on the presence of specific amino acids on the protein surface. They found that human, murine, and hamster interferons can bind to Cu 2+ chelates. Human interferon can bind to a variety of other metal chelates (Co 2+, Ni 2+, and Zn 2+ ) as well. They also studied 5 T. E. Cawston and J. A. Tyler, Biochem. J. 183, 647 (1979). 6 A. R. Torres, E. A. Peterson, W . H . Evans et al., Biochim. Biophys. Acta 576, 385 (1979). 7 H. Kikuchi and M. Watanabe, Anal. Biochem. 115, 109 (1981). 8 L. Sottrup-Jensen, T. E. Petersen, and S. Magnusson, FEBS Lett. 121, 275 (1980). 9 M. F. Scully and V. V. Kakkar, Biochem. Soc. Trans. 9, 335 (1981). l0 I. Ohkubo, T, Kondo, and N. Taniguchi, Biochim. Biophys. Acta 616, 89 (1980). i1 E. Bollin, Jr. and E. Sulkowski, Arch. Virol. 58, 149 (1978). 12 K. Berg and I. Heron, Scand. J. Immunol. 11, 489 (1980). 13 K. C. Chadha, P. M. Grob, A. J. Mikulski et al., J. Gen. Virol. 43, 701 (1979). 14 p. C. P. Ferreira, M. Paucker, R. R. Golgher et al., Arch. Virol. 68, 27 (1981). 15 j. W. Heine, J. van Damme, M. de Ley et al., J. Gen. Virol. 54, 47 (1981). 16 S. Yonehara, Y. Yanase, T. Sano et al., J. Biol. Chem. 256, 3770 (1981). i7 E. Suikowski, K. Vastola, D. Osezek et al., Proc. 4th Int. Syrup. Affinity Chromatogr. Related Techniques Veldhoven, Neth. (T. C. J. Gribnau, J. Visser, and R. J. F. Nivard, eds.), p. 313. Elsevier, Amsterdam, 1981.

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TABLE I REPRESENTATIVE PROTEINS PURIFIED BY M C A C Protein Plasminogen activator lectin CoUagenase Lactoferrin Nonhistone proteins a2-Macroglobulin Human fibrinogen Nucleoside diphosphatase

Dolichos bioflorus

Metal

Ref.

Zn2+ Ca2+ Zn2+ Cu2+ Cu2+

3 4 5 6 7

Zn 2+

8

Zn2+ Zn2+, Cu2+

9 10

the reversibility of binding. It is even possible to resolve different subclasses of human interferon on a Zn 2÷ chelate column.t5 Andersson and Porath 18recently described a method in which immobilized ferric (Fe 3+) ions could be used as a group-specific adsorbant to isolate phosphoproteins and phosphoamino acids. In fact, they could distinguish natural amino acids and their phosphorylated counterparts. A large n u m b e r of amino acids were examined for their affinity to a Fe 3÷ chelate gel. It was found that the majority had very low affinities while the phosphorylated amino acids were tightly bound. When ovalbumin was used as a model o f a phosphoprotein, the matrix was specific enough to resolve the three protein subcomponents (At, A2, and A3) that differed only in their phosphate content. Covalent Chromatography and Bifunctional Agents Although binding of most ligands in affinity chromatography is accomplished through the carboxyl or amino groups (especially on proteins), there are other reactive groups that are available under the proper conditions. The use of these groups may make the separation even more specific than if the carboxyl or amino groups were used. Sulfhydryl-containing matrices can be used to couple proteins containing not only sulfhydryl groups, but also compounds containing C ~ O , C~--C, N~---N, as well as h e a v y metals (e.g., Hg) or alkyl and aryl halides (for a more complete review of this chemistry see Ref. 19). The matrix for this type o f chromatography is made with an active sulfhydryl group that la L. A n d e r s s o n a n d J. Porath, Anal. Biochem. 154, 250 (1986).

~9p. C. Jocelyn, "Biochemistry of the SH Group. The Occurrence, Chemical Properties, Metabolism and Biological Function of Thiols and Disulphides." Academic Press, New York, 1972.

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will form a covalent disulfide bond with the protein of interest. The active group on the matrix is usually a thiopropyl or glutathione moiety. In this type of chromatography a sample or ligand containing thiol groups is bound to the matrix by the formation of a reversible mixed disulfide bond. The use of covalent chromatography is similar to other affinity chromatography procedures. It is a very powerful method for quickly isolating thiol-containing enzymes or specific blood proteins. The flow rate used for the application of the sample is relatively slow, allowing the formation of the disulfide bond. The extent of binding can easily be monitored at 343 nm, indicating the formation of 2-thiopyridone. Buffers should not contain reducing agents [e.g., dithiothreitol (DTT)] since these will interfere with the disulfide formation. After the binding step nonreacting proteins are washed out using the start buffer. A buffer containing a reducing agent or L-cysteine (5-20 mM) at pH 8.0 is then added to the buffer to dissociate the disulfide bond that was formed between the protein (ligand) and the matrix. Higher flow rates can be used in these later steps of the process. One method for regeneration of the column is to prepare a solution of 30-40 mg/ml of 2,2-dipyridyl disulfide in ethanol or 2-propanol. One volume of this solution is mixed with 4 vol of gel in 0.1 M borate buffer, pH 8.0, containing 1 mM EDTA and then refluxed at 80° for 3 hr. The gel is then washed with ethanol and reequilibrated with starting buffer. 2° Caution must be taken regarding the solubilities of materials used for regeneration since several of the reagents are only sparingly soluble in water. Keep in mind when using resins containing active thiol groups that they are able to bind proteins and other components containing heavy metals (e.g., Hg) so bacteriostatic agents containing these moieties should be avoided. Bifunctional reagents also often employ the use of the disulfide bond. These reagents are useful when the KD between the ligand and the sample is very low or the sample is sensitive to extremes o f p H . These agents can be attached to either the ligand or the matrix. The disulfide bond is easily and safely dissociated using reducing agents such as DTT or 2-mercaptoethanol. This allows separation of the sample from the ligand without the use of harsh denaturing agents. Scale-Up After development of a successful analytical separation, scaling up to preparative levels is often desired to produce a larger quantity of a sub2o "Product Data Sheet for Covalent Chromatography." Pharmacia LKB, Piscataway, New Jersey, 1984.

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stance for further study. Scale-up may mean going from microgram to milligram or from milligram to kilogram quantities. The use of high-capacity affinity resins often simplifies the separation by keeping the increase in column volume, and thus elution volume, to a minimum. The general rule of thumb in scaling up is to scale out, not up. This means that the column diameter should be increased while column height remains constant. This is very important. Increasing the column diameter achieves the larger column volume necessary when larger sample volumes are applied. However, by holding the bed height constant, the kinetics involved in the binding of the sample to a ligand will be unaffected. Further, assuming a constant linear flow rate, the residence time of the sample on the column will remain the same. This becomes particularly important since an increase in residence time may lead to changes in the binding characteristics of the compound, or cell, of interest. Conformational changes in a protein, due to column interactions (e.g., electrostatic or hydrophobic) may change the elution pattern. In the most extreme case, elution may become extremely difficult. Maintaining a constant bed height will also limit exposure of both the sample and the matrix to the harsh conditions often required for elution (e.g., acids or chaotropic agents). Flow rate is the next parameter for consideration when scaling up an affinity separation. The linear flow rate (in centimeters per hour, see [29] in this volume) should be maintained in going from a smaller to a larger column. If the linear flow rate is decreased, the residence time of the sample increases. If the flow rate is increased, the kinetics of binding may be shifted in such a way as to prevent adsorption. This will be especially true in cases of weak binding (KD 10-4 to 10 -5 M). The bound molecules are in equilibrium with a small amount of free (unbound) materials. The eluting agent competes with the ligand on the matrix for the free molecules. The rate of elution is limited by the initial dissociation of the bound substance from the gel. Increasing the flow rate will affect this dissociation. Any change in the linear flow rate will change the binding and elution characteristics of a sample with a concomitant change in the resulting separation. The sample is the next parameter to be considered in scaling up. Maintain a constant ratio of sample volume to column volume while maintaining a constant sample concentration. Changes in sample concentration will alter the size and shape of the eluted protein peaks and can have an effect on the binding characteristics (single-point or multipoint attachment) of the sample. Altering the sample-to-column volume ratio can also have an effect in the case of a weak binding interaction. Larger sample

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volumes can potentially lead to coelution of sample with nonadsorbed material. The last important factor in scaling up an affinity chromatography separation is the maintenance of the buffer volume to column volume ratio. The effects of variations in this parameter are evident when elution involves the development of a gradient. If the gradient end points are held constant and the gradient length is increased (i.e., a shallow gradient) broader peaks and greater peak separation may result. Decreasing the length of the gradient (steeper gradient) will sharpen the peaks, but may also reduce separation. Either way, the elution pattern will change. As in all chromatographic procedures other factors, such as the mechanical stability of the matrix, must be considered. The actual physical stability of a gel bead is unchanged by the height or width of the column. However, the maximum flow rate in a packed bed is affected by the column size and is generally reduced as the column length increases. In smaller diameter columns, the walls of the column lend considerable support to the gel bed. The degree of support, and thus the flow rate, varies with the column diameter and height. A matrix can tolerate higher flow rates in smaller columns than in larger ones. When very large columns are used (e,g., >30-cm diameter), the walls no longer offer support to the bed. Consequently, the maximum tolerable flow rate is strictly a function of the matrix stability, and thus will be lower in wider columns. Having a clean sample is just as important when the process is scaled up as when it is first developed in smaller columns. It is advisable to "clean up" the sample before application to the column. In small-scale operations, this is not often done since the matrix can either be easily and quickly cleaned, or it can be disposed of and fresh gel used for the next run. In the case of larger columns, disposal is often too costly and cleaning may not be as expeditious. The pretreatment and clean-up steps used in the initial separation should be incorporated into any scale-up protocol. This clean-up may involve delipidation and/or centrifugation to remove particulates. This keeps the sample composition more consistent during the scale-up. It also helps simplify column maintenance procedures.